Figure 3.
Expression of the IL-12Rβ1 chain and a functional IL-23R complex following downregulation of miR-146b-5p. Purified CLL cells were transfected with miR-146b-5p inhibitor or miR CTR inhibitor, cultured for different times and analyzed for the IL-23R complex expression and for IL-23 production. (A) Time course analysis of the IL-23R complex expression after transfection with miR CTR inhibitor or miR-146b-5p inhibitor in a representative CLL case (GE1-AG114). Cells expressing the IL-23R complex were measured by flow cytometry, determining the simultaneous expression of both the IL-23R and IL-12Rβ1 chains. Double-positive cells were considered as IL-23R complex-positive. Only viable cells were gated, and of these were mainly CD19+CD5+ since cells with this phenotype that were purified before transfection (see also supplemental Figure 7). (B-D)Summary of time course expression of IL23R complex, (C) IL-12Rβ1, and (D) IL-23R side chain determined in cells from 8 CLL cases by flow cytometry before (T0) and after treatment as in (A). Data are expressed as a percentage of positive cells (mean ± SD). (E) IL-23 production in cell supernatants from 6 CLL cases treated as in (A). (F) Representative experiment on cells from GE1-AG114 CLL case to show the presence of a functional IL-23R complex. Purified CLL cells were transfected with the indicated miR inhibitors and cultured for 24 to 72 hours in the presence or absence of IL-23 (100 ng/mL), with/without IL-23-neutralizing mAbs (αIL-23p19). Viable cells (annexin-V/PI-negative cells) were determined after a 72-hour culture. (G) Summary of time course experiments on cells from 8 CLL cases treated and analyzed as in (F). Data are plotted as percent of viable cells mean ± SD, and the P value indicates the differences between the different culture conditions. (H) Determination of cell cycle phases by flow cytometry in CLL cells (GE1-AG114) transfected with the indicated miR inhibitors and cultured for 48 hours in the presence or absence of IL-23 (100 ng/mL), with/without IL-23 neutralizing mAbs (αIL-23p19) in the indicated combinations. Flow logic software was employed for the analyses. Proliferating (G2M) cells are indicated in green. (I) Summary of experiments on cells from 8 different CLL cases performed and analyzed as in (I). The values were determined after 48 hours in culture. P values are indicated (Wilcoxon test). ∗P = .04 and ∗∗P = .0078.

Expression of the IL-12Rβ1 chain and a functional IL-23R complex following downregulation of miR-146b-5p. Purified CLL cells were transfected with miR-146b-5p inhibitor or miR CTR inhibitor, cultured for different times and analyzed for the IL-23R complex expression and for IL-23 production. (A) Time course analysis of the IL-23R complex expression after transfection with miR CTR inhibitor or miR-146b-5p inhibitor in a representative CLL case (GE1-AG114). Cells expressing the IL-23R complex were measured by flow cytometry, determining the simultaneous expression of both the IL-23R and IL-12Rβ1 chains. Double-positive cells were considered as IL-23R complex-positive. Only viable cells were gated, and of these were mainly CD19+CD5+ since cells with this phenotype that were purified before transfection (see also supplemental Figure 7). (B-D)Summary of time course expression of IL23R complex, (C) IL-12Rβ1, and (D) IL-23R side chain determined in cells from 8 CLL cases by flow cytometry before (T0) and after treatment as in (A). Data are expressed as a percentage of positive cells (mean ± SD). (E) IL-23 production in cell supernatants from 6 CLL cases treated as in (A). (F) Representative experiment on cells from GE1-AG114 CLL case to show the presence of a functional IL-23R complex. Purified CLL cells were transfected with the indicated miR inhibitors and cultured for 24 to 72 hours in the presence or absence of IL-23 (100 ng/mL), with/without IL-23-neutralizing mAbs (αIL-23p19). Viable cells (annexin-V/PI-negative cells) were determined after a 72-hour culture. (G) Summary of time course experiments on cells from 8 CLL cases treated and analyzed as in (F). Data are plotted as percent of viable cells mean ± SD, and the P value indicates the differences between the different culture conditions. (H) Determination of cell cycle phases by flow cytometry in CLL cells (GE1-AG114) transfected with the indicated miR inhibitors and cultured for 48 hours in the presence or absence of IL-23 (100 ng/mL), with/without IL-23 neutralizing mAbs (αIL-23p19) in the indicated combinations. Flow logic software was employed for the analyses. Proliferating (G2M) cells are indicated in green. (I) Summary of experiments on cells from 8 different CLL cases performed and analyzed as in (I). The values were determined after 48 hours in culture. P values are indicated (Wilcoxon test). ∗P = .04 and ∗∗P = .0078.

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